Online - 2455-3891 Vol 12, Issue 2, 2019 Print - 0974-2441 Research Article ANTICONVULSANT ACTIVITY OF RAUWOLFIA TETRAPHYLLA LEAF EXTRACT IN SWISS ALBINO MICE AADITYA SINGH1*, SHALINI TRIPATHI2, SINGH PN3 1Department of Pharmaceutical Sciences, Aryakul College of Pharmacy and Research, Lucknow, Uttar Pradesh, India. 2Department of Pharmaceutical Sciences, Rameshwaram Institute of Technology and Management, Uttar Pradesh, India. 3Department of Pharmaceutics, Indian Institute of Technology, BHU, Varanasi, Uttar Pradesh, India. Email: [email protected] Received: 07 July 2018, Revised and Accepted: 01 November 2018 ABSTRACT Objective: Rauvolfia tetraphylla is a plant potentially applicable in Ayurvedic and Unani System of Medicine for the treatment of various diseases. However, the anticonvulsant activity of this plant has not been reported and studied. Therefore, the ethanolic extract of leaf from the plant R. tetraphylla is used to evaluate anticonvulsant activity. Methods: Anticonvulsant activity was screened using maximal electroshock seizure (MES) model and pentylenetetrazole (PTZ)-induced seizure model in Swiss albino mice. The ethanolic extract was also evaluated for rutin and gallic acid content by high-performance thin-layer chromatography studies. Results: Rutin and gallic acid contents were found as 15.60% and 7.81%, respectively. Ethanolic leaf extract (100–800 mg/kg) significantly reduced the duration of seizures which was induced by MES. The same doses also protected animals from PTZ-induced tonic seizures. Conclusion: The study demonstrates that R. tetraphylla plant leaves have significant anticonvulsant activity. Keywords: Anticonvulsant, High-performance thin-layer chromatography, Rauvolfia tetraphylla. © 2019 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ajpcr.2019.v12i2.28334 INTRODUCTION and authenticated by Head, Department of Pharmacognosy and Ethnopharmacology at National Botanical Research Institute, Lucknow, It is now highlighted from literature work that the interest of human India. A voucher specimen no NBRI/RES/RT-20 is preserved for future is continuously increased for the use of natural drugs/phytochemicals reference. The leaves were dried in shade and crushed to obtain coarse obtained from different plant sources for the treatment of various acute powder after authentication. The solvents and chemical were analytical and chronic diseases because of their lesser side effects along with various grade and distilled before use. neurological disorders. Epilepsy is a neurological disorder of the central nervous system which is the third most common disease after stroke Reagents, standards, and drugs and Alzheimer’s [1]. It is a group of related disorders where a person The drug phenytoin sodium was used as standard anticonvulsant suffers from recurrent seizures, an abnormal, disorderly discharging of drug and obtained from Zydus Cadila Healthcare Limited, India. the brain’s nerve cells, resulting in a temporary disturbance of motor, The pentylenetetrazole (PTZ) ( ) was used as standard sensory, or mental function. It is an urgent need to search the medicinal R. tetraphylla plants that can accelerate beneficial effects in seizures. convulsion producing agent purchased from Ranbaxy, India, and the other chemicals such as hexane, ethanol, and dimethylsulfoxide were Rauvolfia belongs to the family Apocynaceae and are well known for also of analytical grade. the presence of indole alkaloids [2]. Rauvolfia tetraphylla L. is native to Mexico, Central America, West Indies, and Northern South America. It has Preparation of extract been cultivated widely as both an ornamental and traditional medicine. It The leaves of R. tetraphylla were powdered and sieved, followed is now utilized throughout to tropics including Australia, Indonesia, China by successive extraction of powdered leaves prepared on the basis and India. Rouwolfia popularly used in Ayurvedic and Unani System of of polarity with hexane and 250 ml of 90% ethanol using Soxhlet Medicines along with a part of folk remedies of most of the Asian countries apparatus at 50°C for 80 h. The process was repeated until complete [3]. It is commonly known as “Be still tree” or “Devil-pepper” and in Hindi extraction and the pooled extract were concentrated under vacuum. as Barachandrika. The alkaloids isolated from the plant material show The resultant extract was filtered (Whatman filter paper No. 1) and was various pharmacological activities such as antipsychotic, antimicrobial, dried/concentrated under reduced pressure using rotator evaporator. anti-inflammatory, anticancer, antihypertensive, antidiarrheal, and The extractive value obtained was approximately 30.12% w/w. The antioxidant [4]. It has been cultivated both as ornamental and traditional extract was stored in cold condition refrigerator throughout the medicine and are available as endangered medicinal plant of Odisha duration of experimentation process. (India) as shrub for the treatment of epilepsy, insomnia, wound, fever, colic, and urinary retention in Ayurveda. The present manuscript High-performance thin-layer chromatography (HPTLC) describes the anticonvulsant efficacy of the ethanolic extract of R. Preparation of working solutions of standard and sample tetraphylla leaf on Swiss albino mice. The working solution of standards (1 mg/ml) and samples (10 mg/ml) was freshly prepared in methanol. The stock solution of standards METHODS 1 mg/mL was diluted in the same solvent to obtain a four working The leaves of R. tetraphylla were collected from the herbal garden solutions in a concentration ranging from 0.1 to 0.4 mg/ml, for of the National Botanical Research Institute, Lucknow, India, calibration. Singh et al. Asian J Pharm Clin Res, Vol 12, Issue 2, 2019, 377-380 HPTLC procedure Convulsions were developed by MES; different phases of the It is well known that HPTLC is used for quantitative analysis, for convulsions (flexor, extensor, convulsion, stupor, and recovery or death) sampling about 10 microliter of sample was applied using Hamilton were observed. Hind-limb tonic extension (HLTE) phase was measured syringe (Camag 100 ml syringe, Switzerland) on a pre-coated plates of as the protection of the convulsion developed by MES model. silica gel, having thickness of 6mm. The sample was spotted 10 mm from the bottom and 15 mm from side of the plate by using Camaglinomat-V Effect of extract on PTZ-induced seizures automated applicator. The applicator is facilitated by nitrogen flow The standard drug PTZ which is used for the generation of seizures providing a delivery speed of 150 ml/s. was administered in a dose of 60 mg/kg i.p 60 min before the test drug, i.e., the ethanolic extract of R. tetraphylla and the parameters such as Development of plates myoclonic, clonic, and tonic–clonic seizures [8] and % protection was The layers were developed in a Camag Twin Trough Glass Chamber, observed. In this model also, Swiss albino mice were divided into five which was presaturated with mobile phase toluene: ethyl acetate: formic groups (n=6): Group I, Group II, Group III, Group IV, and Group V; acid (7:2.5:0.5 v/v). The plate after development was dried and Group I is served as control, Groups II, III, and IV get the ethanolic spraying agent anisaldehyde sulfuric acid was applied for derivatization extract in a dose of 200, 400, or 600 mg/kg, and the Group V served as followed by scanning at 254 nm and 366 nm. The percentage of rutin standard received phenytoin sodium 25 mg/kg. and gallic acid was calculated using the following formula [5]: Statistical analysis The data obtained by different experimental parameters were ()Sample area×standard dilution×purity × 100 statistically evaluated by SPSS software and ANOVA test followed by Standard area×sammple dilution×100 ()student’s t-test (p<0.05) which was used to find the significance. All observations were expressed as mean±standard deviation, and the Experimental animals graph of each data is plotted by GraphPad Prism software. The Swiss albino mice of either sex of body weight (20–30 g) were obtained RESULTS from Central Animal House, Rameshwaram Institute of Pharmacy (Reg. No - 1397/ac/10 Committee for the purpose of Control and Supervision HPTLC of Experimental Animal [CPCSEA]). Swiss albino mice were randomly Quantification of rutin and gallic acid was done by HPTLC; mobile distributed in various groups for the treatment of standard and sample phase used was toluene: ethyl acetate: formic acid (7:2.5:0.5 v/v) which drugs. They were kept at one ambient temperature of 25±1°C and 45– shows very good resolution of spot of the sample against the standards 55% RH in polypropylene cage and acclimatized to laboratory condition on the HPTLC plate. HPTLC plates are visualized in ultraviolet light before a week to start the experiment. The experimental protocols were of 254 nm and 366 nm wavelength. Identification of the presence of approved by the Institutional Animal Ethics Committee constitute under rutin and gallic acid in the extract was performed by comparison of Committee for the Purpose of CPCSEA, Government of India. chromatogram of standard and sample, which is shown to be similar in Fig. 1. By these studies of HPTLC, the amount of rutin and gallic acid is Acute oral toxicity studies 15.60% and 7.81%, respectively (Figs. 2 and 3). The acute toxicity study of an ethanolic extract of R. tetraphylla was done as per the OECD guideline No.420 (OECD, 2002). Swiss MES test albino mice in three different groups of either sex were weighed and placed under standard condition. Method of CPCSEA was adopted for toxicity studies. The extracts were administered in a dose of 50, 300, 1000, and 2000 mg/kg p.o. to different groups of mice each containing 10 animals, and mortality was observed after 25 h. The mortality dose of animals is 2000 mg/Kg. Drug treatment Swiss albino mice were treated both with standardized ethanolic Fig. 1 Picture of leaves of Rauvolfia tetraphylla leaf extract of R.